Increased Amino Acid Absorption Mediated by Lacticaseibacillus rhamnosus IDCC 3201 in High-Protein Diet-Fed Mice

The use of dietary protein products has increased with interests in health promotion, and demand for sports supplements. Among various protein sources, milk protein is one of the most widely employed, given its economic and nutritional advantages. However, recent studies have revealed that milk protein undergoes fecal excretion without complete hydrolysis in the intestines. To increase protein digestibility, heating and drying were implemented; however, these methods reduce protein quality by causing denaturation, aggregation, and chemical modification of amino acids. In the present study, we observed that Lacticaseibacillus rhamnosus IDCC 3201 actively secretes proteases that hydrolyze milk proteins. Furthermore, we showed that co-administration of milk proteins and L. rhamnosus IDCC 3201 increased the digestibility and plasma concentrations of amino acids in a high-protein diet mouse model. Thus, food supplementation of L. rhamnosus IDCC 3201 can be an alternative strategy to increase the digestibility of proteins.


Screening of Probiotic Strain with the Highest Proteolytic Activity
To evaluate the proteolytic activities of probiotic strains, a basal medium containing 0.5% (w/v) tryptone, 0.25% yeast extract, 0.1% glucose, 2.5% skim milk powder, and 1.5% agar was used. Then, 10 μl of each probiotic strain culture (10 8 CFU /ml) was spotted on the agar plate. After incubating the plates at 37°C for 24 h, the length of the clear zone surrounding the colonies was measured using ImageJ software (version 1.8.0, NIH, USA).

Quantification of Compositional Amino Acids in Milk Protein Mixture
For the quantification of amino acids, ninhydrin method was exploited [21]. We employed ion-exchange chromatography to quantify the amino acids in a milk protein mixture comprising whey protein, skimmed goat milk protein, and colostrum protein. The milk protein mixture (0.2 g) was mixed with 10 ml of 6 N HCl in a reactor. After the nitrogen gas injection, the mixture was treated at 110°C for 24 h. The decomposed mixture was concentrated using a vacuum concentrator, adjusted to 50 ml with 0.2 M sodium citrate buffer, and filtered through a 0.45-μm nylon syringe filter. For quantifying 15 amino acids (alanine, arginine, aspartate, glutamate, glycine, histamine, isoleucine, leucine, lysine, phenylalanine, proline, serine, threonine, tyrosine, and valine) except for tryptophan, 20 μl of the filtered sample was injected into Hitachi L-8900 amino acid analyzer with an ultraviolet (UV) detector (Hitachi High-Technologies Corporation, Japan) and an ion-exchange column (#2622PH column 4.6×60 mm). Buffer set for protein hydrolysate, PH-SET KANTO (Hitachi High-Technologies Corporation) which is used to elute amino acids in the ninhydrin method, was used as a mobile phase. Flow rates of the buffer set and ninhydrin buffer were 0.35 and 0.40 ml/min, respectively, and protein peaks were detected at 440 and 570 nm. To quantify tryptophan, high-performance liquid chromatography (Agilent 1260, Agilent Technologies, USA) was performed using a CAPCELL C18 (250 × 4.6 mm, Osaka Soda, Japan) column. As mobile phases, 8.5 mM sodium acetate and methanol (95:5, v/v) were used at a flow rate of 1.0 ml/min. The peak of tryptophan was detected at 280 nm using a UV detector. To quantify methionine and cysteine, the sample was pulverized using a sample grinder and passed through a sieve with a particle size of 500 μm. The sieved sample (2 g) was mixed with acetonitrile and water solution (1:9, v/v; 50 ml), followed by sonication for 20 min at 45°C. The sonicated sample was centrifuged at 3,000 ×g for 20 min. Subsequently, 5 ml of the supernatant was transferred to a 15 ml test tube, and 100 μl of 50% (w/v) KOH was added and stirred for 10 s. After adding 100 μl of 85% (w/v) phosphoric acid to the sample, the supernatant was filtered through a 0.45-μm nylon syringe filter. Next, 20 μl of the sample was injected into a Hitachi L-8900 amino acid analyzer with a UV detector (Hitachi High-Technologies Corporation). An NH 2 column (reversed-phase, 150 mm × 4 mm, Altman Analytik, Germany) was used, and 0.01 M H 3 PO 4 (pH 3.2) and acetonitrile solution (isocratic mode; ratio 77:23, v/v) were employed as the mobile phase at a constant flow rate of 1 ml/min. Finally, the peaks of methionine and cysteine were detected at 214 nm using a UV detector.

Mouse Experiments
Six-week-old male C57BL/6J mice were purchased from Central Lab Animal Inc. (Korea) and maintained in a controlled atmosphere with a 12:12 h light and dark cycle. The mice were fed either a normal diet (ND; 2018S Teklad Global 18% Protein Rodent Diet; Envigo, USA) or a high-protein diet (a mixture of ND and milk protein blends; 6:4 w/w) for eight weeks. Mice (n = 24) were randomly divided into four groups: 1) ND; 2) High-protein diet (HPD); 3) HPD + L. rhamnosus IDCC 3201 (HPD + LLrh; 10 7 CFU/day); 4) HPD + L. rhamnosus IDCC 3201 (HPD + HLrh; 10 8 CFU/day). ND or HPD were supplied ad libitum, and L. rhamnosus IDCC 3201 was orally administered to mice daily. Animal studies were approved by the Institutional Animal Care and Use Committee of the Chungbook National University (approval number: CBNUA-1687-22-02). The body weight, and water and food consumption were measured weekly. After eight weeks, mice were subjected to a 6 h fasting period and anesthetized with diethyl ether. Blood samples were collected from the abdominal veins and centrifuged at 600 ×g for 20 min. The serum was collected and stored at −70°C until further use. To analyze free amino acids in serum, we employed a Hitachi L-8900 amino acid analyzer with a UV detector (Hitachi High-Technologies Corporation, Japan) and an ion-exchange column (#2622PH column 4.6 × 60 mm).

Statistical Analyses
Results are expressed as mean ± standard deviation (SD). For statistical data comparisons, analysis of variance (ANOVA) analysis followed by Duncan test and Student's t-test were performed using STATISTICA version 7 (TIBCO Software Institute, USA).

Screening of Lacticaseibacillus rhamnosus IDCC 3201 with Higher Proteolytic Activity
To select an optimal probiotic strain capable of improving milk protein digestion, the proteolytic activities of L. rhamnosus IDCC 3201, S. thermophilus IDCC 2201, and E. faecium IDCC 2102 were evaluated. Each probiotic strain was spotted on a basal agar medium containing skim milk and incubated for 24 h to measure the size of the halo produced by proteolysis. In results, L. rhamnosus IDCC 3201 produced an 8.18 ± 0.78 mm halo surrounding the colony, whereas S. thermophiles IDCC 2201 and E. faecium IDCC 2102 produced halos of 3.47 ± 0.35 and 3.29 ± 0.22 mm, respectively (Fig. 1A). The L. rhamnosus IDCC 3201 generated halo via proteolysis of skim milk was 2.4-and 2.5-fold larger than those generated by S. thermophiles IDCC 2201 and E. faecium IDCC 2102, respectively (Fig. 1B). Considering pea protein, L. rhamnosus IDCC 3201 also exhibited the highest proteolytic activity among examined strains (Fig. 1B). In order to investigate the cause of the higher proteolytic activity of L. rhamnosus IDCC 3201 beyond the other probiotic strains, the genes of protease, peptidase, and proteinase of representative L. rhamnosus strains, L. rhamnosus NCTC 13764, L. rhamnosus ATCC 53103, and L. rhamnosus IDCC 3201 were investigated ( Table 1). As a result, the genome of L. rhamnosus NCTC 13764 contained 13 proteases, 38 peptidases, and 2 proteinases, the genome of L. rhamnosus ATCC 53103 contained 12 proteases, 40 peptidases, and 2 proteinases, and the genome of L. rhamnosus IDCC 3201 contained 13 proteases, 40 peptidases, and 2 proteinases. In particular, L. rhamnosus IDCC 3201 had serine protease/ABC transporter B family protein tagC, probable endopeptidase YddH, and Beta-Ala-His dipeptidase, which are absent in other strains.
During the eight-week experimental period, we observed no significant difference in the food efficiency ratio (body weight gain (g)/food intake (g) × 100) between examined groups. To determine whether the co-administration of L. rhamnosus IDCC 3201 and the milk protein mixture would enhance protein digestibility in HPD diet-fed mice, plasma concentrations of amino acids were quantified using an amino acid analyzer. Firstly, the HPD group showed significantly increased aspartate and glutamate absorption than the ND group (Fig. 3). Next, coadministration of L. rhamnosus IDCC 3201 and milk protein mixture resulted in significant increased serum concentration of glycine, proline, and tryptophan in both HPD + LLrh and HPD + HLrh groups by up to 27.47, 54.01, and 64.92%, respectively, compared to those in the HPD group. Meanwhile, aspartate, glutamate, serine and taurine concentrations were only elevated in HPD + HLrh group by 23.59, 60.45, 36.95, and 47.29%, respectively, compared to those in the HPD group (Table 3).

Discussion
Based on the notion that intestinal microbes degrade dietary proteins and in turn this promotes the intestinal amino acid absorption in the host [7], this study investigated whether co-administration of probiotics and proteins would increase serum amino acid concentration in high-protein diet-fed (HPD) mice. In this study, Lacticaseibacillus rhamnosus IDCC 3201 was selected as a potential probiotic bacterium that can increase amino acid absorption in the host (Fig. 1). Genome analysis indicates that this strain has specific proteases and  Each value is presented as mean ± standard deviation (SD). 1 Normal diet (ND) group 2 High-protein diet (HPD) group 3 High-protein diet (HPD) and orally administered L. rhamnosus IDCC 3201 (10 7 CFU/day; LLrh) 4 High-protein diet (HPD) and orally administered L. rhamnosus IDCC 3201 (10 8 CFU/day; HLrh) *Significant at p < 0.05; **significant at p < 0.01; ***significant at p < 0.001 compared with high-protein diet group.
peptidases. Especially, serine proteases are thought to be main enzymes of protein degradation, because their signaling molecules for extracellular secretion are a member of triggering and cleaving family of proteinaseactivated receptors (Table 1) [22]. Furthermore, the proteolytic enzymes such as PepO, IspA, and RseP were founded only in L. rhamnosus IDCC 3201, compared to other rhamnosus strains. PepO has distinct cleavage site for a s1 -casein fragment 1-23, and the protease IspA, which is frequently found mainly in the Bacillus species, is crucial role in stationary phase, where cell growth being to stop [24,25]. Lastly, RseP stimulates transcriptional factor of σ E extra-cytoplasmic stress response and in turn, eliminates signal peptides of extracelluar proteins in the secondary processing in cytoplasmic membrane [26]. On the other hands, The PepR and PepX which are prolinespecific peptidases are found in only other L. rhamnosus strains, while PepXP (prolyl dipeptidyl aminopeptidase) are only found in in L. rhamnosus IDCC3201 and these genes are frequently found in Lactococcus lactis strains [27,28]. Milk protein mixture used in this study is mainly composed of glutamate (17.6%, w/w), leucine (10.5%), aspartate (10.3%), and lysine (9.0%) ( Table 1), and this protein composition is similar to the previously reported milk protein mixture, mainly composed of leucine, glutamate, aspartate, and proline [29]. In consistent with amino acid composition of the tested protein mixture, HPD-animal experiments showed significantly increased serum glutamate and aspartate, compared to normal diet group (Fig. 3). Furthermore, co-administration of Lacticaseibacillus rhamnosus IDCC 3201 and milk protein mixture increased 7 amino acid absorption of in highprotein diet-fed (HPD) mice: aspartate, glutamate, serine, glycine, taurine, proline, and tryptophan. Among the essential amino acids, only tryptophan concentration increased up to 4.6-fold in co-administration group, compared to HPD group, whereas among the branched-chain amino acids, there was no significantly increased amino acid (Table 2).
Participants in clinical trials who ingested 25 g of a milk protein and 10 9 CFU of B. coagulans GBI-30 had increased concentrations of arginine, isoleucine, serine, ornithine, and methionine in their serum, compared to those who ingested only protein [14]. Moreover, ingestion of a 20g of plant protein and 10 billion CFU of probiotics consisting of L. paracasei LP-DG (CNCM I-1572) and L. paracasei LPC-S01 (DSM 26760) improved serum concentration of methionine, histidine, valine, leucine, and isoleucine [13]. Together with these results, coingestion of either (both) probiotic(s) or (and) protein changes of composition of microbiota in the intestine. For example, high protein diet led to an increase in the proportion of Bifidobacterium spp. and Lactobacillus spp., whereas, ingestion of Bacillus spp. increased the population of healthy gut-related bacteria [30].
Amino acids are crucial for growth development, and health of the host, exerting various regulatory functions in cells including protein synthesis, cell signaling, metabolic regulations, and immune function [31]. With regards to immune function, aspartate and glutamate play versatile roles in the metabolism of leucocytes and lymphocytes [32]. Serine is crucial for several cellular functions, including neurotransmission, folate and methionine cycles, and sphingolipid synthesis [33]. Glycine, synthesized from serine, is a biosynthetic intermediate for protein (e.g., collagen) and is an inhibitory neurotransmitter in the central nervous system [34]. Taurine, synthesized from methionine and cysteine metabolism, is essential for various physiological functions, including glycolysis, glycogenesis, osmoregulation, anti-oxidation, and detoxification [35]. Proline mediates key functions in protein function and the regulation of cellular redox homeostasis [36]. Lastly, tryptophan can be converted into 3idolepropionic acid (IPA), indole-3-aldehyde (I3A), and indole by gastrointestinal microbes: IPA functions as a neuroprotectant; I3A maintains mucosal reactivity; and indole associated with vascular and chronic kidney diseases [37].
Collectively, our finding suggests that the co-administration of L. rhamnosus IDCC 3201 and dietary proteins confer health benefits in terms of amino acid absorption through intestinal enterocytes.